Ferrata Storti Foundation Unraveling the cellular origin ...€¦ · assess the association between...

1176 haematologica | 2019; 104(6)

Received: September 7, 2018.

Accepted: December 20, 2018.

Pre-published: January 24, 2019.

©2019 Ferrata Storti FoundationMaterial published in Haematologica is covered by copyright.All rights are reserved to the Ferrata Storti Foundation. Use ofpublished material is allowed under the following terms andconditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. Copies of published material are allowed for personal or inter-nal use. Sharing published material for non-commercial pur-poses is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode,sect. 3. Reproducing and sharing published material for com-mercial purposes is not allowed without permission in writingfrom the publisher.

Correspondence: PABLO MENÉNDEZ [email protected]

IGNACIO [email protected]

Haematologica 2019Volume 104(6):1176-1188

ARTICLE Acute Lymphoblastic Leukemia

doi:10.3324/haematol.2018.206375

Check the online version for the most updatedinformation on this article, online supplements,and information on authorship & disclosures:www.haematologica.org/content/104/6/1176

Ferrata Storti Foundation

B-cell acute lymphoblastic leukemia is the commonest childhood cancer.In infants, B-cell acute lymphoblastic leukemia remains fatal, especiallyin patients with t(4;11), present in ~80% of cases. The pathogenesis of

t(4;11)/KMT2A-AFF1+ (MLL-AF4+) infant B-cell acute lymphoblastic leukemiaremains difficult to model, and the pathogenic contribution in cancer of thereciprocal fusions resulting from derivative translocated-chromosomesremains obscure. Here, “multi-layered” genome-wide analyses and validationwere performed on a total of 124 de novo cases of infant B-cell acute lym-phoblastic leukemia uniformly diagnosed and treated according to theInterfant 99/06 protocol. These patients showed the most silent mutationallandscape reported so far for any sequenced pediatric cancer. Recurrent muta-tions were exclusively found in K-RAS and N-RAS, were subclonal and werefrequently lost at relapse, despite a larger number of non-recurrent/non-silentmutations. Unlike non-MLL-rearranged B-cell acute lymphoblasticleukemias, B-cell receptor repertoire analysis revealed minor, non-expandedB-cell clones in t(4;11)+ infant B-cell acute lymphoblastic leukemia, and RNA-sequencing showed transcriptomic similarities between t(4;11)+ infant B-cellacute lymphoblastic leukemias and the most immature human fetal liverhematopoietic stem and progenitor cells, confirming a “pre-VDJ” fetal cellularorigin for both t(4;11) and RASmut. The reciprocal fusion AF4-MLL wasexpressed in only 45% (19/43) of the t(4;11)+ patients, and HOXA clustergenes are exclusively expressed in AF4-MLL-expressing patients. Importantly,AF4-MLL/HOXA-expressing patients had a significantly better 4-year event-free survival (62.4% vs. 11.7%, P=0.001), and overall survival (73.7 vs. 25.2%,P=0.016). AF4-MLL expression retained its prognostic significance when ana-lyzed in a Cox model adjusting for risk stratification according to theInterfant-06 protocol based on age at diagnosis, white blood cell count andresponse to prednisone. This study has clinical implications for disease out-come and diagnostic risk-stratification of t(4;11)+ infant B-cell acute lym-phoblastic leukemia.

Unraveling the cellular origin and clinicalprognostic markers of infant B-cell acute lymphoblastic leukemia using genome-wideanalysis Antonio Agraz-Doblas,1,2 Clara Bueno,2# Rachael Bashford-Rogers,3# AninditaRoy,4,# Pauline Schneider,5 Michela Bardini,6 Paola Ballerini,7 GianniCazzaniga,6 Thaidy Moreno,1 Carlos Revilla,1 Marta Gut,8,9 Maria G. Valsecchi,10

Irene Roberts,4,11 Rob Pieters,5 Paola De Lorenzo,10 Ignacio Varela,1,$,* PabloMenendez2,12,13,$,* and Ronald W. Stam5

1Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, Santander, Spain; 2Josep Carreras Leukemia Research Institute-Campus Clinic,Department of Biomedicine, School of Medicine, University of Barcelona, Spain;3Department of Medicine, University of Cambridge, Cambridge Biomedical Campus, UK;4Department of Paediatrics, University of Oxford, UK; 5Princess Maxima Center for PediatricOncology, Utrecht, the Netherlands; 6Centro Ricerca Tettamanti, Department of Pediatrics,University of Milano Bicocca, Fondazione MBBM, Monza, Italy; 7Pediatric Hematology, A.Trousseau Hospital, Paris, France; 8CNAG-CRG, Center for Genomic Regulation, Barcelona,Spain; 9Universitat Pompeu Fabra, Barcelona, Spain; 10Interfant Trial Data Center, Universityof Milano-Bicocca, Monza, Italy; 11MRC Molecular Haematology Unit, MRC WeatherallInstitute of Molecular Medicine, University of Oxford, UK; 12Instituciò Catalana de Recerca iEstudis Avançats (ICREA), Barcelona, Spain and 13Centro de Investigación Biomédica enRed de Cáncer (CIBERONC), ISCIII, Barcelona, Spain#These authors contributed equally to this work.$These senior authors contributed equally to this work.

ABSTRACT

Introduction

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent cancer in children.1 Current 5-year survival rates in pediatric BCP-ALL approach 90%.However, BCP-ALL in infants (iBCP-ALL; <1 year of age)remains clinically challenging with an aggressive earlyclinical presentation in uniquely vulnerable hosts.2Approximately 80% of iBCP-ALL are diagnosed withchromosomal rearrangements involving the mixed-lin-eage leukemia (KMTA2, also called MLL) gene, locatedon 11q23,3–5 which confers a dismal prognosis especiallyin patients carrying the t(4;11)/KMT2A-AFF1+ (MLL-AF4+).6–8MLL is a H3K4 histone methyltransferase required for

normal hematopoiesis and HOX gene expression.9,10Leukemia transformation by MLL fusions requires therecruitment of the H3K79 histone methyltransferaseDot1L to the MLL transcriptional complex.11,12 Indeed, anH3K79 methylation profile defines both mouse andhuman t(4;11)/MLL-AF4+ BCP-ALL.13 Importantly, MLLrearrangements (MLLr) occur prenatally during embryon-ic/fetal hematopoiesis, and the concordance rate foriBCP-ALL in identical twins with a monochorionic pla-centa is close to 100%.14–17 This, coupled to the extremelyshort latency, suggests that MLL fusions might be suffi-cient for leukemogenesis.4 Accordingly, genome-widestudies using both single nucleotide polymorphismarrays and whole-genome sequencing revealed thatMLLr iBCP-ALL has a very low frequency of somaticmutations with the predominant clone carrying ~1.3non-silent mutations and one copy number alteration.18–20 Although these studies were performed at low cover-age sequencing they reinforce the concept that MLLriBCP-ALL requires few additional mutations to inducefull transformation. In contrast, MLL-AF4-induced leuke-mogenesis has proven difficult to model.4,9 With theexception of a recent work by Lin et al.21,22 who fusedhuman MLL to murine Af4, creating an artificial leuke-mogenic human-mouse chimeric fusion, current murineand humanized models of MLL-AF4+ BCP-ALL do notfaithfully recapitulate the disease pathogenesis/pheno-type, suggesting that MLL-AF4 per se is insufficient to ini-tiate leukemogenesis.23–28The few mutations and copy number alterations pres-

ent in MLLr iBCP-ALL seem subclonal and not alwaysretained at relapse.20 Intratumor heterogeneity drivesclonal evolution in response to microenvironmental cuesand cytotoxic treatment and therefore recurrent muta-tions at diagnosis and relapse may be found in minor butclinically relevant subclones.29 Here we aimed to addressthe clinical relevance of subclonal mutations and geneexpression signatures in a large cohort of iBCP-ALL. Todo this, we performed deeper exome sequencing alongwith whole-genome DNA- and RNA-sequencing on alarge cohort of 50 MLLr and non-MLL iBCP-ALL patientsuniformly treated and followed up according to anInterfant treatment protocol.30 Similarly to Anderson etal.,20 we report a silent mutational landscape in iBCP-ALLirrespective of the MLL rearrangement/status. However,strikingly, our genome-wide DNA and RNA analysesrevealed new, clinically relevant information about dis-ease outcome and cell-of-origin for t(4;11) and RASmutations.

Methods

PatientsBone marrow or peripheral blood samples from 124 infants

(<12 months old) diagnosed with either pro-B or pre-B-cell ALLwere used in this study. The discovery cohort of patients wascomposed of 42 de novo cases: 27 with the t(4;11) encoding forMLL-AF4, five with the t(9;11) encoding for KMT2A-MLLT3(MLL-AF9) and ten without MLLr (non-MLL B-other BCP-ALLwithout numerical or structural chromosomal abnormalitiesreported at diagnosis). Additionally, for eight MLL-AF4+ iBCP-ALLpatients matched diagnostic-relapse samples were available allow-ing for longitudinal studies. MLL rearrangements were confirmedby fluorescence in-situ hybridization.31,32 For validation, an addi-tional cohort of patients, comprising 43 MLL-AF4+, 11 MLL-AF9+,and 28 non-MLL iBCP-ALL cases, was used. All patients wereenrolled in the Interfant99 treatment study. Bone marrow sampleswere collected at Erasmus MC-Sophia Children’s Hospital(Rotterdam, the Netherlands), Armand Trousseau Hospital (Paris,France), and San Gerardo Pediatric Hospital (Monza, Italy).Complete remission bone marrow samples were available for allpatients. The clinical and genetic features of the patients are pre-sented in Online Supplementary Table S1. As a control for the RNA-sequencing studies, CD34+CD19+ healthy B-cell progenitors werepurified by fluorescence-activated cell sorting (FACS) from 22-week old human fetal livers (FL) as previously described.32 FLhematopoietic stem and progenitor cells (HSPC) were processedand FACS-purified from second trimester human FL as previouslydescribed.33 Briefly, cells were processed and stained for flowcytometry with up to ten fluorophore-conjugated monoclonalantibodies [antibodies (clone): CD34PECy7 (8G12), CD45RAFITC (HI100), CD19APC (HIB19), CD123PE (9F5), CD90 PECy5(5E10), CD38 Pacific blue (HIT2), lineage cocktail APC (CD2(RPA-2.10)/CD3 (OKT3)/CD14 (61D3)/ CD16(CB16)/ CD19(HIB19)/CD56 (TULY56)/CD235a (HIR2)]. FACS was performedusing a BD FACSAria II (Becton Dickinson). Gates were set withunstained and fluorescence minus one controls, on viable cells.Data were analyzed using FlowJo software (Tree Star). Gatingstrategies are as described in the results section. The study wasapproved by the Barcelona Clinic Hospital (2013/8529) andHammersmith and Queen Charlotte’s Hospital (04/Q0406/145)research ethics committees.

Statistical analysisFor quantitative variables, a one-tailed t-test was used to identi-

fy significant differences between groups. For qualitative vari-ables, a Fisher exact test was used in order to identify significantdifferences between groups of patients. Software for analysis ofmutations and gene expression have their own statistical modelsexplained in detailed in the references. Where multiple tests wereperformed the significance is shown corrected for multiple testing.Mutation allele frequency evolution was plotted with the R pack-age distribution Fishplot. Patterns Fisher exact test was used toassess the association between clinical characteristics and pres-ence of RAS mutations or AF4-MLL expression. Event-free sur-vival was defined as time from diagnosis to first event, i.e. resist-ance, relapse, death from any cause, or second malignant neo-plasm. Observation periods were censored at the time of last con-tact when no events were reported. Event-free survival curveswere estimated with the Kaplan-Meier method and standarderrors (SE) were calculated according to Greenwood. Differencesin event-free survival and overall survival between groups werecompared with the log-rank test. Analysis of the prognostic rele-vance of AF4-MLL/HOXA expression in combination with risk

Cellular origin and clinical prognostic markers of infant MLLr B-ALL

haematologica | 2019; 104(6) 1177

stratification according to the Interfant-06 protocol (based on ageat diagnosis, white blood cell count and response to prednisone)was performed with the Cox model and the Wald test. All testswere two-sided. Analyses were performed using SAS 9.2.

DNA, RNA and B-cell receptor (VDJ) repertoiregenome-wide analyses and data analysis

Preparation and analysis of all DNA and RNA genome-widehigh-throughput sequencing is detailed in the OnlineSupplementary Methods, Online Supplementary Figure S1 and OnlineSupplementary Table S2.

Results

At diagnosis infant B-cell precursor acute lymphoblastic leukemia shows a silent mutationallandscape irrespective of MLL gene statusWhole-exome sequencing and whole-genome sequenc-

ing analyses showed a silent mutational landscape in thethree iBCP-ALL subtypes studied here: MLL-AF4+, MLL-AF9+ and non-MLL (n=42 patients, Online SupplementaryTable S1). Our study revealed an average of one genomicrearrangement and 2.5 non-silent single nucleotide vari-ants, a 2-fold higher number than that reported by

Andersson et al.,20 likely reflecting the 3-fold largersequencing coverage (Figure 1A, Online SupplementaryFigure S1 and Online Supplementary Table S3). All mutationsfound at diagnosis were validated using orthogonal meth-ods. This mutational frequency is the lowest described forany other pediatric tumor type according to recentreports34 (Online Supplementary Figure S2). Intriguingly, onethird of the mutations validated showed a mutant allelefrequency (MAF) <20% indicating that iBCP-ALL containsgenetically different intratumoral subclones despite itsgenomic stability, likely explaining the higher mutationalload than that reported by Andersson et al.20 (Figure 1Aand Online Supplementary Table S3). Despite the paucity ofmutations, ~80% of the validated protein-coding muta-tions (90/116) are predicted to produce deleterious effectson the protein (Online Supplementary Figure S3A) whichmight support a strong selective pressure in iBCP-ALL. Togain insights into the molecular mechanisms underlyingthe accumulation of mutations, we analyzed the enrich-ment of specific mutational signatures as described byAlexandrov et al.35 In the MLL-AF4+ iBCP-ALL subgroupwe identified a significant enrichment of signature 1 char-acterized by the accumulation of C>T/G>A transitions,linked to a spontaneous deamination of 5-methylcytosine(Online Supplementary Figure S3B,C).35 This mutational sig-

A. Agraz-Doblas et al.

1178 haematologica | 2019; 104(6)

Figure 1. Somatic mutations detected by whole-exome sequencing in the discovery cohort of infant B-cell precursor acute lymphoblastic leukemia. (A) Total num-ber of mutations identified in each individual patient. The total number of non-synonymous mutations (yellow area, right Y axis) and mutant allele frequency (MAF)for each mutation (individual dots, left Y axis) are represented. (B) Oncodrive software identified the PI3K-RAS pathway as the only recurrently mutated pathway ininfant B-cell precursor acute lymphoblastic leukemia. The distribution of mutations in genes of the PI3K-RAS pathway is shown for all patients within the three iBCP-ALL subgroups: [total 42 patients: 27 t(4;11)+, 5 t(9;11)+ and 10 MLLwt].

A

B

nature has also been described in other pediatric tumors,suggesting that iBCP-ALL is not subjected to a specificmutational signature.We also determined the molecular breakpoint of all

MLLr at the base-pair level. In t(4;11)/MLL-AF4+ iBCP-ALL, the AF4 breakpoints were almost invariably localizedwithin intron 3 whereas MLL breakpoints were foundbetween introns 9 and 11 (Online Supplementary Table S4).3We found whole-genome sequencing reads compatiblewith an AF4-MLL reciprocal rearrangement in all samples(Online Supplementary Figure S4). AF4-MLL genomic break-points were validated by polymerase chain reaction capil-lary sequencing and they were located nearby MLL-AF4breakpoints, confirming a reciprocal chromosomaltranslocation.

RAS-PI3K is the only recurrently mutated pathway in infant B-cell precursor acute lymphoblasticleukemia with NRAS mutations being significantlymore frequent in t(4;11)+ patientsDespite the low number of mutations found per sample,

38% of the sequenced iBCP-ALL patients displayed acti-vating/gain-of-function mutations in either KRAS orNRAS. Additional mutations in other genes members ofthe RAS-PI3K pathway such as FGFR4, JAK2, PTPN11,SETD2, or FLT3were also identified (Figure 1B). To furthervalidate the unique recurrence of KRAS and NRAS muta-tions, we performed targeted sequencing of these muta-tions in a large, additional validation cohort of infantpatients (n=82) and confirmed that 34% of the iBCP-ALLcases carry mutations in either KRAS or NRAS36 (Figure2A). Interestingly, the overall frequency of RAS mutationsdiffered slightly between the different cytogenetic sub-groups of iBCP-ALL, with the MLL-AF4+ subgroup show-ing the highest frequency (42%) and the MLL-AF9+ sub-group the lowest (19%). This difference was basicallyattributed to the frequency of NRAS mutations, whichwas 6-fold more common in the MLL-AF4+ subgroup

(32% vs. 6%, Fisher exact test P=0.01) (Figure 2A,B).Surprisingly, we observed that many iBCP-ALL patients

had mutations in both KRAS and NRAS, or more than one(different) mutation in the same gene (Figure 2A,C). Tofurther analyze the biological contribution of KRAS andNRAS mutations, we calculated the MAF of individualmutations and observed that the majority of patients whohad a single RAS mutation (either KRAS or NRAS) hadMAF scores between 0.20 and 0.45, suggesting that themutation is present in a major leukemic subclone(P=0.0025). By contrast, those patients harboring two ormore RAS mutations displayed MAF scores between 1%and 20%, compatible with these RAS mutations being indistinct and smaller leukemic subclones. We then ana-lyzed the impact of RAS mutations on disease outcomeand found no clinical correlation of RAS mutations witheither clinical outcome (overall survival, event-free sur-vival, central nervous system infiltration) or diagnosticparameters (gender, age, percentage of blasts and whiteblood cells) (Online Supplementary Figure S5).

Evidence of clone selection and genomic instability at relapsePaired diagnostic-relapse samples were available for

eight MLL-AF4+ iBCP-ALL patients, permitting longitudi-nal studies. Whole-exome sequencing revealed an 8-foldincrease in the number of somatic non-synonymousmutations at relapse (19.5 mutations/patient, range:1-434,paired t-test P=0.03) (Figure 3A,B and Online SupplementaryTable S3). We performed orthogonal validation for 160random mutations, and 90% and 75% of mutations withMAF >15% and <15%, respectively, were confirmed (datanot shown). Similarly to diagnosis, the majority of thesomatic mutations found at relapse had MAF commonly<30%, suggesting the existence of multiple leukemic sub-clones (Figure 3A). Importantly, none of the new de novosomatic mutations found at relapse was found in morethan one patient, likely reflecting an intrinsic genomic


haematologica | 2019; 104(6) 1179

Figure 2. Frequent somatic mutations in RAS genes in both the discovery and validation cohorts. (A) Specific KRAS and NRAS mutations recurrently found in eachpatient by high coverage targeted sequencing. (B) Proportions of patients with mutations in KRAS (brown), NRAS (yellow) or both (gray) within the three infant B-cellprecursor acute lymphoblastic leukemia subgroups. (C) Mutant allele frequency of KRAS (brown squares) or NRAS (yellow circles) mutations in each individualpatient. Discovery cohort, n=42; validation cohort, n=82.

A

B C

instability of leukemic clones surviving induction/consoli-dation chemotherapy. This is further reflected by a signif-icant enrichment of signature 6 associated with defectiveDNA mismatch repair, including a higher number of smallindels, observed in MLL-AF4+ patients at relapse (OnlineSupplementary Figure S6A).To delineate the evolutionary clonal structure from diag-

nosis to relapse, we performed high-coverage targetedsequencing on the identified mutations in paired diagnos-tic-remission-relapse samples.37 Importantly, the mainleukemic clone at relapse was always present at diagnosisalthough in some cases with a very low MAF, suggestinga chemotherapy-induced clonal pressure selecting forresistant/adapted leukemic subclones (Figure 3C).


1180 haematologica | 2019; 104(6)

Figure 3. Clonal evolution andgenomic instability at relapse. (A)Total number of mutations identifiedfor each patient in paired diagnostic-relapse samples. Total number ofnon-synonymous mutations (yellowarea, right Y axis) and mutant allelefrequency (MAF) for each mutation(individual dots, left Y axis) are repre-sented for paired diagnostic andrelapsed (R) samples. (B) Circos plotrepresentation of the total number ofmutations identified at diagnosis andrelapse for a representative patient(MA4_17). Genomic rearrangementsare represented by lines connectingboth breakpoints. Copy number alter-ations (blue=gains, red=losses) arerepresented by the outer gray circle.Somatic mutations (both singlenucleotide variants and indels) aredepicted in the center of the circleand the affected gene is indicated. (C)Graphic representation of clonal evo-lution in paired diagnostic (DX)-relapsed (RL) samples. The number ofunique somatic mutations called atdiagnosis (orange), relapse (yellow) orshared between DX and REL (red) areindicated. Bigger gene names indicatehigher MAF for the mutations sharedat DX and REL. (D) Dynamics of RAS-mutated clones identified as MAF inmatched DX-Remission-REL trios(n=8).

A

B

C

D

Interestingly, we found a correlation between the numberof mutations and time to relapse in MLL-AF4+ patients,with a trend towards a higher mutational load in patientswith late relapses (Online Supplementary Figure S6B). Wenext analyzed the clonal evolution of RAS-mutatedleukemic clones at relapse. We found that the contribution

of the RAS mutations varied among patients: one-third ofthe iBCP-ALL patients had RAS-mutated clones at relapse(MA4_20 and MA4_22 increased the size of the RAS-mutated initial clone and in MA4_14 a de novo RAS muta-tion emerged), whereas it was lost in two-thirds of thepatients (MA4_17, MA4_18, MA4_23, MA4_24) (Figure


haematologica | 2019; 104(6) 1181

Figure 4. Transcriptional signature ofinfant B-cell precursor acute lym-phoblastic leukemia samples. (A)Heatmap representing FLT3, PROM1,MEIS1 and HOXA gene expressionaccording to the infant B-cell precur-sor acute lymphoblastic leukemia(iBCP-ALL) cytogenetic group and RASmutations. (B) Top panel: heatmapshowing HOXA cluster gene expres-sion according to the expression ofthe reciprocal fusion AF4-MLL.Bottom panel: quantitative poly-merase chain reaction validating highexpression of HOXA cluster genes int(4;11) iBCP-ALL patients expressingAF4-MLL. (C,D) Four-year event-freesurvival (C) and overall survival (D)Kaplan-Meier curves for t(4;11) iBCP-ALL patients according to AF4-MLLexpression, n=43 t(4;11)+ patients.(E) Heatmap representation of select-ed genes for the signaling pathwaysmost significantly deregulated. Rightpanels represent positive pathwayenrichment called by gene set enrich-ment analysis software. Total 42patients: 27 t(4;11)+, 5 t(9;11)+ and10 MLLwt.

A B

C

D

E

3D). This indicates that infants with MLL-AF4+ BCP-ALLrelapse irrespective of the status of KRAS and NRAS.Thus, subclones carrying KRASmutations do not exert anadvantage over non-mutated clones, despite representinga recurrent genetic insult at diagnosis. Hence, this wouldargue against a leukemia-initiating role for RASmutations.38 Alternatively, RAS mutations might indeedbe leukemogenic drivers, but the treatment-inducedgenetic instability observed at relapse may compensate denovo RASmutations, acting as new leukemia drivers coop-erating with MLL-AF4 during relapse.

HOXA cluster genes are only expressed in t(4;11)+patients expressing the reciprocal fusion AF4-MLLwhich determines clinical outcomeTo gain insights into the mechanisms underlying leuke-

mogenesis in these mutationally silent MLLr and MLLgermline iBCP-ALL patients, we performed RNA-sequencing in the discovery cohort of patients (n=42)using FL-derived CD34+CD19+ healthy B-cell progenitorsas controls, as these cells most likely represent the healthycounterparts of the leukemic blast stalled at the pro/pre-B-cell differentiation stage. We first surveyed the expressionof the genes previously reported to be specific to eitherMLLr iBCP-ALL or specifically to MLL-AF4+ iBCP-ALL39.RNA-sequencing profiling confirmed that these genes seg-regate patients according to the molecular subtype, MLL-AF4+, MLL-AF9+ and MLL germline (Online SupplementaryFigure S7). We also observed, at diagnosis, a strong upreg-ulation of the MLL target genes FLT3,40 MEIS1, PROM1and HOXA genes in many of our MLLr iBCP-ALL samplesbut not in MLL germline samples (t-test, P<0.05) (Figure4A), thus validating our RNA-sequencing approach. Strikingly, the reciprocal AF4-MLL fusion gene was dis-

cernibly expressed in 19/43 (45%) of the t(4;11)+ iBCP-ALL samples, and its expression was always maintained atrelapse (data not shown). We then compared the genes dif-ferentially expressed between AF4-MLL-expressing andnon-expressing t(4;11)+ patients and found a striking posi-tive correlation between the expression of the HOXA genecluster and overexpression of the reciprocal AF4-MLLfusion (t-test, P=0.002) (Figure 4B). These AF4-MLL/HOXA-expressing patients (n=19) had a significantlybetter prognosis than those lacking AF4-MLL/HOXAexpression (n=24). Four-year event-free and overall sur-vival rates were 62.4% (SE, 11.3%) versus 11.7% (SE,10.2%) (P=0.001) (Figure 4C), and 73.7% (SE, 10.1%) ver-sus 25.2% (SE, 10.3%) (P=0.016) (Figure 4D), respectively.When “AF4-MLL expression” was analyzed in a Coxmodel adjusting for risk stratification (medium risk or highrisk according to the Interfant-06 protocol based on age atdiagnosis, white blood cell count and response to pred-nisone), it retained its prognostic significance with a haz-ard ratio for patients lacking AF4-MLL expression of 3.42[95% confidence interval (95% CI): 1.35-8.63; P=0.01)compared to those expressing AF4-MLL/HOXA, whilerisk group was not significant (HR for high risk vs. medi-um risk, 1.34; 95% CI: 0.59-3.03; P=0.49). This is the firststudy showing that AF4-MLL overexpression correlatesvery well with transcriptional deregulation of the HOXAgene cluster in iBCP-ALL and that the co-expression ofAF4-MLL and HOXA gene cluster identifies a subgroup oft(4;11)+ iBCP-ALL with a very more favorable clinical out-come.We next explored new molecular pathways involved in

the pathogenesis of iBCP-ALL, by performing an unbiasedtranscriptional analysis of the RNA-sequencing data fromthe iBCP-ALL patients. We found deregulated expressionof a total of 3,905 genes, of which 2,575 (66%) wereupregulated and 1,330 (34%) downregulated as comparedwith those of healthy FL-derived B-cell progenitors, illus-trating the global transcriptional activation nature of MLLfusions (Online Supplementary Figure S8).25,41 Furthermore, asignificant upregulation of genes involved in the control ofcell growth, including the CDK inhibitors P21, P16, P19,P27 and components of the transforming growth factor-bpathway such as TGFB1, SMAD and ACVR1B, wasobserved in iBCP-ALL (Figure 4D and OnlineSupplementary Figure S9). By contrast, iBCP-ALL showed arobust downregulation of genes involved in DNA integri-ty checkpoints such as CHEK1, CHEK2, ATM, ATR andRAD17, and in double-strand break repair genes includingERCC4, BRCA1, POLA1 and RAD51 (Figure 4E and OnlineSupplementary Figure S9). These transcriptional changeswere validated by quantitative reverse transcriptase poly-merase chain reaction in ten patients per group (OnlineSupplementary Figure S10). Deregulation of DNA integritycheckpoints and double-strand break repair genes maywell contribute to the genomic instability observed atrelapse, and might explain the enrichment in C>T/G>Atransitions, associated with the spontaneous deaminationof 5-methylcytosine (Online Supplementary Figures S3 andS6).By using FL-derived normal B-cell progenitors as con-

trols, differences between leukemic blasts and their nor-mal counterparts could be identified but this does notallow the definition of transcriptomic differences withinthe iBCP-ALL cytogenetic groups. We, therefore, analyzedthe RNA-sequencing data comparing the genes differen-tially expressed in MLL-AF4+ versus MLL-AF9+ and MLL-wildtype iBCP-ALL patients, without considering normalB-cell progenitors as controls. A gene ontology analysis(gene set enrichment analysis, GSEA) performed with thegenes differentially expressed revealed that MLL-AF4+

patients show, as compared to both MLL-AF9+ and MLL-wildtype patients, a significant upregulation of genes asso-ciated with cellular catabolism, coupled to a significantdownregulation of negative regulators of the PI3-MAPKpathway, as well as of genes involved in lymphoid differ-entiation and RNApol II transcriptional regulation (Figure5). This suggests, respectively, a metabolic change in MLL-AF4+ cells towards rapid energy generation while reinforc-ing the basal hyperactivation of the PI3-MAPK pathwayby RASmutations (Figures 1 and 2), a poorly differentiatedcellular origin of t(4;11), and an impairment of the normalfunction of AF4, a key component of the RNApol II tran-scription complex.

Deep-sequencing analysis of B-cell receptor repertoires suggests a hematopoietic stem cell/earlypre-VDJ progenitor as the cell-of-origin for t(4;11) and RAS mutationsWe next analyzed BCR repertoires to gain insights into

the immunoglobulin heavy chain (IgH) rearrangementclonal composition of paired diagnostic-relapse samplesfrom t(4;11)+ iBCP-ALL (4 pairs). BCR are generatedthrough DNA recombination during B-cell differentiationand represent unique markers for each B-cell clone.Because the BCR sequence provides a molecular tag foreach B-cell clone, high-throughput sequencing of BCR


1182 haematologica | 2019; 104(6)

provides a detailed analysis of B-cell population dynamicsand clone tracking.42,43 BCR sequencing was therefore per-formed to address whether t(4;11)+ iBCP-ALL cellsexpressed fully rearranged BCR from which increased lev-els of B-cell clonal expansion may be observed and todetermine whether there are detectable levels of B-cellclonal persistence over time indicative of B-cell clonal sur-vival. BCR sequencing was performed on t(4;11)/MLL-AF4+ iBCP-ALL peripheral blood samples (blasts >98%)using a polymerase chain reaction-based method37 withadditional incorporation of unique molecular barcodes,allowing for accurate quantitation of relative B-cell clonefrequency. After BCR sequence filtering, each sampleyielded between 1,583-46,863 BCR (1,213-38,426 uniqueBCR) (Online Supplementary Table S5).We first delineated the relative clonality in these

patients, and found that the BCR repertoires from t(4;11)+patients did not exhibit significantly expanded VDJ-rearranged B-cell clones (Figure 6A) either at diagnosis orrelapse compared to healthy peripheral blood samples

(Figure 6B). This is in contrast to non-MLL BCP-ALLpatients (n=5) including three patients with t(1;19)/TCF3-PBX1 (EF2-PBX1), one with t(12;21)/ETV6-RUNX1 (TEL-AML1) and one with t(9;22)/BCR-ABL1, which were allfound to be significantly clonal, with large B-cell clonescomprising ~3-40% of total BCR (Figure 6B,C).37 Giventhe persistence of both t(4;11) and RASmutations in MLL-AF4+ iBCP-ALL, the lack of B-cell clonal expansion or per-sistence supports the model that t(4;11)/MLL-AF4+ iBCP-ALL malignant cells are developmentally stalled at the pro-B stage, and that the cellular origin of such genomic driv-ers has to be pre-VDJ stem/progenitor cells. Finally, in order to understand whether the fetal cell-of-

origin in iBCP-ALL lies upstream of committed B progen-itors, we compared the transcriptome of iBCP-ALL blasts(n=42) with that of highly purified human FL HSPC pop-ulations (3-7 for each population) (Figure 7A,B and OnlineSupplementary Table S6) by RNA-sequencing. In keepingwith the results of the BCR analysis, our principal compo-nent analysis revealed a gene expression signature for


haematologica | 2019; 104(6) 1183

Figure 5. Specific transcriptionaldifferences between MLL-AF4+

and MLL-AF9+ or MLLwt infant B-cell precursor acute lymphoblas-tic leukemia patients. Here, FL-derived CD34+CD19+ progenitorswere not included as normalizersin the analysis in order to avoidpotential bias. Gene set enrich-ment analysis (GSEA) was per-formed with the genes differen-tially expressed between MLL-AF4+ patients and MLL-AF9+ orMLLwt patients. MLL-AF4+ infantB-cell precursor acute lym-phoblastic leukemia (iBCP-ALL)patients showed a significantoverexpression of genes associ-ated with cellular catabolism,coupled to a significant downreg-ulation of negative regulators ofthe PI3-MAPK pathway, as wellas of genes involved in lymphoiddifferentiation and RNApol IItranscriptional regulation ascompared to both MLL-AF9+ andMLLwt iBCP-ALL patients. Thebottom panels represent positivepathway enrichment called byGSEA software. Total 42patients: 27 t(4;11)+, 5 t(9;11)+

and 10 MLLwt.

primitive Lin-CD34+CD38-CD19- FL HSPC (hematopoieticstem cells, multipotent progenitors, and lymphoid-primedmultipotent progenitors, which lie upstream of B progen-itors) very similar to t(4;11)+ iBCP-ALL, while FL-commit-ted B progenitors clustered as a transcriptionally differententity (Figure 7C).

Discussion

We set out to perform multi-layered sequencing on alarge cohort iBCP-ALL patients, all enrolled in the interna-tional, collaborative Interfant treatment protocol. The factthat all patients were identically treated provides legitima-cy and confidence in potential correlations of clinicalvalue. Our study revealed an average of 2.5 non-silent sin-gle nucleotide variants, a 2-fold higher number than thatreported by Andersson et al.,20 likely reflecting the 3-foldlarger sequencing coverage. This silent mutational land-scape, even in non-MLL iBCP-ALL, likely reflects the veryyoung age of these patients, reinforcing the notion thatinfant cancer is a developmental disease with not enoughtime to develop somatic mutations. We also found theonly recurrent, but subclonal, mutations occur in theKRAS and NRAS genes (gain-of-function mutations),although the frequency of subclonal NRAS mutations is

significantly higher in t(4;11)+ patients. In line with ourprevious work we found no recurrent mutations in theFLT3 gene.40Analysis of clonal evolution of RAS-mutated clones

from diagnosis to relapse revealed that one-third of thepatients still carry RAS mutations at relapse, whereas theother two-thirds of patients who relapse have lost thediagnostic RAS mutation. This is in accordance withrecently published data by Trentin et al.,36 and suggeststhat the therapy is able to eliminate the RAS-mutatedclone in some patients, while in other patients the RASmutation seems to confer chemoresistance, allowing theseclones to evade treatment.44 Intriguingly, ~25% of thepatients carry more than one RAS-mutated clone at diag-nosis, indicating a selection bias towards mutations in theRAS genes, or activated RAS pathways during leukemictransformation. From this perspective, the occurrence ofpatients carrying multiple distinct clones with activatedRAS pathways may point to convergent evolution ofclones capable of controlling the proliferation rate.However, arguing against this is the substantial represen-tation of patients not carrying RAS mutations at all.Hence, the role of RAS mutations in t(4;11)+ iBCP-ALLremains obscure, and the available data suggest that RASpathway mutations are unlikely leukemia-initiatinglesions. Indeed, Tamai et al.45 showed that leukemogenesis


1184 haematologica | 2019; 104(6)

Figure 6. Analysis of B-cellreceptor repertoires suggest ahematopoietic stem cell/earlypre-VDJ progenitor as the cell-of-origin for t(4;11)/MLL-AF4+

infant B-cell precursor acutelymphoblastic leukemia. (A)Cloud-plots of B-cell receptor(BCR) repertoires from two rep-resentative t(4;11)+ infant B-cellprecursor acute lymphoblasticleukemia (iBCP-ALL) patientsdepicting the existence of manyminor non-expanded B-cellclones either at diagnosis orrelapse. Each vertex representsa unique BCR sequence, andthe relative vertex size is propor-tional to the number of identicalreads. (B) Largest BCR clonesize in t(4;11)+ iBCP-ALL,healthy individuals and non-t(4;11)+ pediatric BCP-ALL. (C)Cloud-plots of BCR repertoiresof representative t(1;19)/E2A-PBX1+, t(12;21)/TEL-AML1+ andt(9;22)/BCR-ABL+ patientsshowing high clonality of B-cellclones. The samples from theiBCP-ALL patients who wereBCR-sequenced were four MLL-AF4+ diagnostic-relapse pairs,three E2A-PBX1+ samples, oneTEL-AML1+ sample and oneBCR-ABL+ sample.

A B

C

of transgenic mice expressing human MLL-AF4 could besignificantly accelerated by KRAS mutations. However,although activated KRAS did cooperate with MLL-AF4 inhuman cord blood-derived CD34+ HSPC to promoteextramedullary infiltration and central nervous systeminfiltration it failed to initiate leukemia in engrafted mice.27Importantly, we report a lack of correlation between RAS

status and parameters associated with diagnosis or diseaseoutcome such as overall survival, event-free survival, cen-tral nervous system infiltration, gender, percentage ofblasts and white blood cells and age, further supportingthe concept that RAS mutations are not leukemia-initiat-ing/propagating lesions.Clearly, this brings us back to the central question of


haematologica | 2019; 104(6) 1185

Figure 7. Comparison of the transcriptome of human fetalCD34+ hematopoietic stem and progenitor cell populations toinfant B-cell precursor acute lymphoblastic leukemia. (A)Schematic representation of B-cell development in human fetalliver (FL) showing immunophenotypic definitions for hematopoi-etic stem cells (HSC), multipotent progenitors (MPP), lymphoid-primed multipotent progenitors (LMPP), committed B progeni-tors (CBP) and B cells. The onset and expected patterns of IgHrearrangements59,60 are depicted as red arrows. (B) Sorting strat-egy for FL hematopoietic stem and progenitor (HSPC) popula-tions by fluorescence-activated cell sorting. The sorting gates foreach population are shown in representative flow plots on theleft. The purity of the sorted populations is depicted on the rightdemonstrating >95% purity. (Lin, Lineage cocktail). (C) Principalcomponent analysis of gene expression of infant B-cell precursoracute lymphoblastic leukemia (iBCP-ALL) samples (n=42) and FLHSPC populations (n=3-7) using the top 1,000 variablyexpressed genes, as determined by RNA-sequencing. FL HSPCas in (A); MAF4, MLL-AF4+ iBCP-ALL; MA9, MLL-AF9+ iBCP-ALL;MLLwt, MLL wildtype iBCP-ALL.

A

B

C

whether or not MLL-AF4 by itself is sufficient to initiateBCP-ALL in humans. The silent mutational landscapeobserved in this study and by others20 certainly votes infavor of MLL-AF4+ iBCP-ALL being initiated by a single“big-bang” transformation hit, probably in a short-livedbut highly proliferative prenatal B-cell progenitor.4 Thishypothesis is supported by recent work by Lin et al., whoindeed demonstrated that enforced expression of a fusiontranscript consisting of human MLL and murine Af4 incord blood-derived CD34+ HPSC is sufficient to inducepro-B ALL in xenografted immunodeficient mice.21,22 Yet,similar results using a human MLL-AF4 transcript remainto be established.Although MLL-AF4 by itself may be sufficient to induce

BCP-ALL without significant contributions from coopera-tive genetic lesions, the contribution of the MLL-AF4 andRAS mutations to leukemogenesis should take intoaccount the nature of both the fetal target cell for transfor-mation and the leukemia-initiating cell, according to theincreasingly accepted stochastic stem cell model of B-ALL.46,47 Here, we employed high-throughput BCR-sequencing of the IgH locus to delineate the dynamics ofclonality of B-cell populations in paired diagnosis-relapsesamples of t(4;11)/MLL-AF4+ iBCP-ALL. While pediatricpatients with E2A-PBX1+, TEL-AML1+ and BCR-ABL1+ B-ALL all had significantly clonal disease, with a major VDJrearranged B-cell IgH clone accounting for up to 40% of allBCR, infants with MLL-AF4+ BCP-ALL exhibited a BCRrepertoire composed of thousands of minor, non-expandedVDJ rearranged IgH B-cell clones. Because MLL fusions areclonal and RAS mutations are found in clones of relativebig size, this suggests that MLL fusions with or withoutRASmutations are likely to originate in primitive fetal pro-genitors that have a germline or an incompletelyrearranged (DJ) IgH locus.48 Indeed, an unsupervised com-parison of the transcriptome of FL HSPC populations andiBCP-ALL blasts suggests that while the gene expression ofprimitive FL HSPC (Lin-CD38-CD34+CD19- populations) issimilar to that of iBCP-ALL, FL B progenitors(CD34+CD19+) are transcriptionally distinct. Our data ele-gantly reinforces previous fluorescence in-situ hybridiza-tion findings suggesting that a primitive “pre-VDJ”stem/progenitor cell (perhaps CD34+CD19-) may representthe cell in which both t(4;11) and RASmutations arise.14,31,49Cooperative leukemogenic events in iBCP-ALL may

need to be sought beyond genetic insults; for instance, epi-genetic and transcriptomic deregulation. MLL-AF4 mightonly induce BCP-ALL in cells that meet certain epigeneticand transcriptomic make-up criteria, either influenced bymicroenvironmental cues, or characteristic of the cell-of-origin.31 Indeed, lesions such as RAS mutations may con-tribute to disease pathogenesis only against certain intrin-sic epigenetic or transcriptomic backgrounds present inthe cell in which the MLL translocations occurred50,51. Thisis supported by the limited impact of RAS mutations intranscriptomic signatures associated with leukemia origin,development and pathogenesis, although this is likely dueto the subclonal nature of RAS mutations.38 However, inline with the reported contribution of RAS mutations toextramedullary infiltration of MLLr BCP-ALL blasts,27RAS-mutated patients displayed a transcriptomic signa-ture associated with migration.The functional and molecular contribution of the recip-

rocal fusion genes resulting from the derivative translo-cated chromosomes remains obscure in cancer. The AF4-

MLL genomic fusion was previously detected in 80-85%of t(4;11)+ patients.5,52 Our “multi-layered omics”approach allowed for the exact characterization of thet(4;11) molecular DNA/RNA break points and the identi-fication of those patients expressing the reciprocal AF4-MLL fusion. We now report that the AF4-MLL reciprocalfusion is expressed in only 50% of t(4;11)+ iBCP-ALLpatients. Strikingly, there was a previously unrecognizedand very significant positive correlation between theupregulation of the HOXA gene cluster and the expres-sion of AF4-MLL. Of note, a recent study showed thatapproximately half of t(4;11)+ patients do not have anactivated HOXA signature.44,53,54 Furthermore, in therecent MLL-Af4-induced B-ALL xenograft model MLL-Af4 failed to bind to HOXA genes and therefore HOXAgene expression was not upregulated.21 This is experi-mentally supported by chromatin immunoprecipitation-sequencing analysis performed in human embryonic stemcells transduced with MLL-AF4, AF4-MLL or both show-ing a significant enrichment of H3K79 methylated regionsspecifically associated with HOX-A cluster genes in dou-ble fusion-expressing hematopoietic derivatives, estab-lishing a functional and molecular cooperation betweenMLL-AF4 and AF4-MLL fusions during humanhematopoietic development (data not shown). Strikingly,AF4-MLL-expressing patients had a 5-fold longer event-free survival and a 3-fold longer overall survival comparedto t(4;11)+ iBCP-ALL patients lacking AF4-MLL expres-sion, which is in line with previous reports suggestingthat high HOXA gene expression is associated withimproved survival and lower risk of relapse.22,39 Becausethe expression of AF4-MLL is not analyzed in routinemolecular diagnosis, our “multi-layered omics” approachwas critical to unraveling the association between AF4-MLL and HOXA expression, thus identifying a novel sub-group of t(4;11)+ iBCP-ALL with better clinical outcome.It is very important for routine diagnostic and clinicalpractice that when the expression of AF4-MLL was eval-uated in a Cox model adjusting for risk stratification(medium risk or high risk according to the Interfant-06protocol), it retained its prognostic significance.Mechanistically, AF4-MLL contains the SET domain dis-

rupted from its "specification domain", the N-terminalportion of MLL, which binds to MEN1 and LEDGF thusshaping the gene-targeting module of the MLL gene.When AF4-MLL is expressed, the N-terminal portion issubstituted by the AF4 N-terminus (AF4N) which is thecrucial domain for binding to and strongly activating RNApolymerase II (RNAP II) for transcriptional elongation.Thus, expression of A4M-MLL may induce robust RNAPII-dependent gene transcription by overwriting the elon-gation control process in a dominant fashion.55–58 Wehypothesize that a likely function of AF4-MLL could be toprepare the ground for MLL-AF4 or other transcriptionfactors to skew normal and leukemic hematopoietic cellfate decisions. This also explains why MLL-AF4, but notAF4-MLL, seems to be necessary in 100% of patients. Despite being a developmental cancer, iBCP-ALL

patients did not show reactivation of pluripotent orembryonic-like gene expression signatures as revealed byRNA-sequencing. Additional research is required to deci-pher the nature of the insults initiating MLLr iBCP-ALL, asso far we can only speculate on the data currently avail-able. Whole-genome pyrosequencing will likely provideunique insights into the DNA methylome landscape of


1186 haematologica | 2019; 104(6)

this mutationally silent iBCP-ALL. This study has clinicalimplications in the diagnostic risk-stratification of t(4;11)+iBCP-ALL.

AcknowledgmentsWe would like to thank the Santander Supercomputing Service

for IT support. The human fetal material was provided by theJoint MRC/Wellcome Trust (grant# MR/R006237/1) HumanDevelopmental Biology Resource (http://hdbr.org). This workwas supported by the European Research Council (CoG-2014-646903 to PM; and StG-2014-637904 to IV), the SpanishMinistry of Economy and Competitiveness (SAF-SAF2013-43065 to PM and SAF2016-76758-R to IV), the Asociación

Española Contra el Cáncer (AECC-CI-2015), FEROFoundation, and the ISCIII (PI14-01191) to CB. PM/IV alsoacknowledge financial support from The Obra Social La Caixa-Fundaciò Josep Carreras, The Inocente Inocente Foundation,Fundación Ramón Areces and The Generalitat de Catalunya(SGR330). IR was supported by a Programme Grant fromBloodwise (LLR 13001) and by the Oxford NIHR BiomedicalCentre based at Oxford University Hospitals NHS Trust andUniversity of Oxford. PM is an investigator of the Spanish CellTherapy cooperative network (TERCEL). AR was supported bya Clinician Scientist Fellowship from Bloodwise (14041). Thiswork was motivated by our patients and it honors the vital exam-ple given to us by the family of AMC.


haematologica | 2019; 104(6) 1187

References

1. Pui C-H, Evans WE. A 50-year journey tocure childhood acute lymphoblasticleukemia. Semin Hematol. 2013;50(3):185–196.

2. Pui CH, Mullighan CG, Evans WE, RellingMV. Pediatric acute lymphoblastic leukemia:where are we going and how do we getthere? Blood. 2012;120(6):1165–1174.

3. Meyer C, Burmeister T, Gröger D, et al. TheMLL recombinome of acute leukemias in2017. Leukemia. 2018;32(2):273–284.

4. Sanjuan-Pla A, Bueno C, Prieto C, et al.Revisiting the biology of infant t(4;11)/MLL-AF4+ B-cell acute lymphoblastic leukemia.Blood. 2015;126(25):2676–2685.

5. Marschalek R. Mechanisms of leukemogen-esis by MLL fusion proteins. Br J Haematol.2011;152(2):141–154.

6. Ribeiro RC, Pui CH. Prognostic factors inchildhood acute lymphoblastic leukemia.Hematol Pathol. 1993;7(3):121–142.

7. Pieters R, Schrappe M, De Lorenzo P, et al. Atreatment protocol for infants younger than1 year with acute lymphoblastic leukaemia(Interfant-99): an observational study and amulticentre randomised trial. Lancet.2007;370(9583):240–250.

8. Biondi A, Cimino G, Pieters R, Pui C-H.Biological and therapeutic aspects of infantleukemia. Blood. 2000;96(1):24–33.

9. Milne TA. Mouse models of MLL leukemia:recapitulating the human disease. Blood.2017;129(16):2217–2223.

10. Nakamura T, Mori T, Tada S, et al. ALL-1 isa histone methyltransferase that assembles asupercomplex of proteins involved in tran-scriptional regulation. Mol Cell. 2002;10(5):1119–1128.

11. Chen CW, Armstrong SA. Targeting DOT1Land HOX gene expression in MLL-rearranged leukemia and beyond. ExpHematol. 2015;43(8):673–684.

12. McLean CM, Karemaker ID, van Leeuwen F.The emerging roles of DOT1L in leukemiaand normal development. Leukemia.2014;28(11):2131–2138.

13. Krivtsov AV, Feng Z, Lemieux ME, et al.H3K79 methylation profiles define murineand human MLL-AF4 leukemias. Cancer.2009;14(5):355–368.

14. Greaves MF, Maia AT, Wiemels JL, Ford AM.Leukemia in twins: lessons in natural histo-ry. Blood. 2003;102(7):2321–2333.

15. Ford AM, Ridge SA, Cabrera ME, et al. Inutero rearrangements in the trithorax-related

oncogene in infant leukaemias. Nature.1993;363(6427):358–360.

16. Gale KB, Ford AM, Repp R, et al.Backtracking leukemia to birth: identifica-tion of clonotypic gene fusion sequences inneonatal blood spots. Proc Natl Acad Sci U SA. 1997;94(25):13950–13954.

17. Bueno C, Montes R, Catalina P, RodríguezR, Menendez P. Insights into the cellular ori-gin and etiology of the infant pro-B acutelymphoblastic leukemia with MLL-AF4rearrangement. Leukemia. 2011;25(3):400–410.

18. Bardini M, Galbiati M, Lettieri A, et al.Implementation of array based whole-genome high-resolution technologies con-firms the absence of secondary copy-numberalterations in MLL-AF4-positive infant ALLpatients. Leukemia. 2011;25(1):175–178.

19. Dobbins SE, Sherborne AL, Ma YP, et al. Thesilent mutational landscape of infant MLL-AF4 pro-B acute lymphoblastic leukemia.Genes Chromosom Cancer. 2013;52(10):954–960.

20. Andersson AK, Ma J, Wang J, et al. The land-scape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias.Nat Genet. 2015;47(4):330–337.

21. Lin S, Luo RT, Ptasinska A, et al. Instructiverole of MLL-fusion proteins revealed by amodel of t(4;11) pro-B acute lymphoblasticleukemia. Cancer Cell. 2016;30(5):737–749.

22. Lin S, Luo RT, Shrestha M, Thirman MJ,Mulloy JC. The full transforming capacity ofMLL-Af4 is interlinked with lymphoid line-age commitment. Blood. 2017;130(7):903–907.

23. Yamamoto H. Successful sustained engraft-ment after reduced-intensity umbilical cordblood transplantation for adult patients withsevere aplastic anemia. Blood. 2011;116(26):6123–6132.

24. Montes R, Ayllón V, Prieto C, et al. Ligand-independent FLT3 activation does not coop-erate with MLL-AF4 to immortalize/trans-form cord blood CD34+ cells. Leukemia.2014;28(3):666–674.

25. Bueno C, Montes R, Melen GJ, et al. Ahuman ESC model for MLL-AF4 leukemicfusion gene reveals an impaired earlyhematopoietic-endothelial specification.Cell Res. 2012;22(6):986–1002.

26. Bueno C, Ayllón V, Montes R, et al. FLT3activation cooperates with MLL-AF4 fusionprotein to abrogate the hematopoietic spec-ification of human ESCs. Blood.2013;121(19):3867–3878.

27. Prieto C, Stam RWRW, Agraz-Doblas A, et

al. Activated KRAS cooperates with MLL-AF4 to promote extramedullary engraftmentand migration of cord blood CD34+ HSPCbut is insufficient to initiate leukemia.Cancer Res. 2016;76(8):2478–2489.

28. Krivtsov A V., Armstrong SA. MLL translo-cations, histone modifications andleukaemia stem-cell development. Nat RevCancer. 2007;7(11):823–833.

29. Varela I, Menendez P, Sanjuan-Pla A.Intratumoral heterogeneity and clonal evolu-tion in blood malignancies and solid tumors.Oncotarget. 2017;8(39):66742–66746.

30. Driessen E, Lorenzo P, M C, et al. Outcomeof congenital acute lymphoblastic leukemiatreated on the Interfant-99 protocol. Blood.2009;114(18):3764–3768.

31. Menendez P, Catalina P, Rodríguez R, et al.Bone marrow mesenchymal stem cells frominfants with MLL-AF4+ acute leukemia har-bor and express the MLL-AF4 fusion gene. JExp Med. 2009;206(13):3131–3141.

32. Muñoz-López A, Romero-Moya D, PrietoC, et al. Development refractoriness of MLL-rearranged human B cell acute leukemias toreprogramming into pluripotency. Stem CellReports. 2016;7 (4):602–618.

33. Roy A, Cowan G, Mead AJ, et al.Perturbation of fetal liver hematopoieticstem and progenitor cell development by tri-somy 21. Proc Natl Acad Sci U S A.2012;109(43):17579–17584.

34. Gröbner SN, Worst BC, Weischenfeldt J, etal. The landscape of genomic alterationsacross childhood cancers. Nature. 2018;555(7696):321-327.

35. Alexandrov LB, Nik-Zainal S, Wedge DC, etal. Signatures of mutational processes inhuman cancer. Nature. 2013;500(7463):415–421.

36. Trentin L, Bresolin S, Giarin E, et al.Deciphering KRAS and NRAS mutatedclone dynamics in MLL-AF4 paediatricleukaemia by ultra deep sequencing analy-sis. Sci Rep. 2016;6:34449.

37. Bashford-Rogers RJM, Nicolaou KA,Bartram J, et al. Eye on the B-ALL: B-cellreceptor repertoires reveal persistence ofnumerous B-lymphoblastic leukemia sub-clones from diagnosis to relapse. Leukemia.2016;30(12):2312–2321.

38. Prelle C, Bursen A, Dingermann T,Marschalek R. Secondary mutations int(4;11) leukemia patients. Leukemia.2013;27(6):1425–1427.

39. Stam RW, Schneider P, Hagelstein JAP, et al.Gene expression profiling-based dissectionof MLL translocated and MLL germline

acute lymphoblastic leukemia in infants.Blood. 2010;115(14):2835–2844.

40. Chillón MC, Gómez-Casares MT, López-Jorge CE, et al. Prognostic significance ofFLT3 mutational status and expression levelsin MLL-AF4 and MLL-germline acute lym-phoblastic leukemia. Leukemia. 2012;26(11):2360–2366.

41. Boelens JJ, Aldenhoven M, Purtill D, et al.Outcomes of transplantation using varioushematopoietic cell sources in children withHurler syndrome after myeloablative condi-tioning Key Points. Blood. 2013;121(10):3981–3987.

42. Bashford-Rogers RJM, Palser AL, Huntly BJ,et al. Network properties derived from deepsequencing of human B-cell receptor reper-toires delineate B-cell populations. GenomeRes. 2013;23(11):1874–1884.

43. Bruggemann M, Schrauder A, Raff T, et al.Standardized MRD quantification inEuropean all trials: Proceedings of theSecond International Symposium on MRDassessment in Kiel, Germany, 18-20September 2008. Leukemia. 2010;24(3):521–535.

44. Driessen EMC, van Roon EHJ, Spijkers-Hagelstein JAP, et al. Frequencies and prog-nostic impact of RAS mutations in MLL-rearranged acute lymphoblastic leukemiain infants. Haematologica. 2013;98(6):937–944.

45. Tamai H, Miyake K, Takatori M, et al.Activated K-Ras protein accelerates humanMLL/AF4-induced leukemo-lympho-mogenicity in a transgenic mouse model.Leukemia. 2011;25(5):888–891.

46. Elder A, Bomken S, Wilson I, et al. Abundant

and equipotent founder cells establish andmaintain acute lymphoblastic leukaemia.Leukemia. 2017;31(12):2577–2586.

47. Prieto C, López-Millán B, Roca-Ho H, et al.NG2 antigen is involved in leukemia inva-siveness and central nervous system infiltra-tion in MLL-rearranged infant B-ALL.Leukemia. 2018;32(3):633–644.

48. Jansen MWJC, Corral L, van der Velden VHJ,et al. Immunobiological diversity in infantacute lymphoblastic leukemia is related tothe occurrence and type of MLL generearrangement. Leukemia. 2007;21(4):633–641.

49. Hotfilder M, Röttgers S, Rosemann A, et al.Leukemic stem cells in childhood high-riskALL/t(9;22) and t(4;11) are present in primi-tive lymphoid-restricted CD34+CD19-cells.Cancer Res. 2005;65(4):1442–1449.

50. Bergmann AK, Castellano G, Alten J, et al.DNA methylation profiling of pediatric B-cell lymphoblastic leukemia with KMT2Arearrangement identifies hypomethylationat enhancer sites. Pediatr Blood Cancer.2017;64(3):e26251.

51. Malouf C, Ottersbach K. The fetal liver lym-phoid-primed multipotent progenitor pro-vides the prerequisites for the initiation oft(4;11) MLL-AF4 infant leukemia.Haematologica. 2018 Jun 14. [Epub ahead ofprint]

52. Kowarz E, Burmeister T, Lo Nigro L, et al.Complex MLL rearrangements in t(4;11)leukemia patients with absent AF4MLLfusion allele. Leukemia. 2007;21(6):1232–1238.

53. Trentin L, Giordan M, Dingermann T, BassoG, Te Kronnie G, Marschalek R. Two inde-

pendent gene signatures in pediatric t(4;11)acute lymphoblastic leukemia patients. Eur JHaematol. 2009;83(5):406–419.

54. Kühn A, Löscher D, Marschalek R. TheIRX1/HOXA connection: insights into anovel t(4;11)- specific cancer mechanism.Oncotarget. 2016;7(23):35341–35352.

55. Wilkinson AC, Ballabio E, Geng H, et al.RUNX1 is a key target in t(4;11) leukemiasthat contributes to gene activation throughan AF4-MLL complex interaction. Cell Rep.2013;3(1):116–127.

56. Benedikt A, Baltruschat S, Scholz B, et al.The leukemogenic AF4-MLL fusion proteincauses P-TEFb kinase activation and alteredepigenetic signatures. Leukemia. 2011;25(1):135–144.

57. Scholz B, Kowarz E, Rössler T, Ahmad K,Steinhilber D, Marschalek R. AF4 and AF4Nprotein complexes: recruitment of P-TEFbkinase, their interactome and potential func-tions. Am J Blood Res. 2015;5(1):10–24.

58. Mück F, Bracharz S, Marschalek R. DDX6transfers P-TEFb kinase to the AF4/AF4N(AFF1) super elongation complex. Am JBlood Res. 2016;6(3):28–45.

59. van Zelm MC, van der Burg M, de Ridder D,et al. Ig gene rearrangement steps are initiat-ed in early human precursor B cell subsetsand correlate with specific transcription fac-tor expression. J Immunol. 2005;175(9):5912–5922.

60. Böiers C, Richardson SE, Laycock E, et al. Ahuman IPS model implicates embryonic B-myeloid fate restriction as developmentalsusceptibility to B acute lymphoblasticleukemia-associated ETV6-RUNX1. DevCell. 2018;44(3):362–377.e7.


1188 haematologica | 2019; 104(6)

Ferrata Storti Foundation Unraveling the cellular origin ...€¦ · assess the association between...

Documents

Transcript of Ferrata Storti Foundation Unraveling the cellular origin ...€¦ · assess the association between...